Methods and compositions for inhibition of angiogenesis with EM-138

ABSTRACT

The present invention comprises a group of compounds that effectively inhibit angiogenesis. More specifically, thalidomide and various related compounds such as thalidomide precursors, analogs, metabolites and hydrolysis products have been shown to inhibit angiogenesis and to treat disease states resulting from angiogenesis. Importantly, these compounds can be administered orally.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional of application Ser. No.08/950,673, filed Oct. 16, 1997, which is a continuation-in-part of U.S.patent application Ser. No. 08/025,046, filed Mar. 1, 1993.

TECHNICAL FIELD

[0002] The present invention relates to methods and compositions forpreventing unwanted angiogenesis in a human or animal. Moreparticularly, the present invention relates to a method for preventingunwanted angiogenesis, particularly in angiogenesis dependent orassociated diseases, by administration of compounds such as thalidomideand related compounds.

BACKGROUND OF THE INVENTION

[0003] As used herein, the term “angiogenesis” means the generation ofnew blood vessels into a tissue or organ. Under normal physiologicalconditions, humans or animals only undergo angiogenesis in very specificrestricted situations. For example, angiogenesis is normally observed inwound healing, fetal and embryonal development and formation of thecorpus luteum, endometrium and placenta. The control of angiogenesis isa highly regulated system of angiogenic stimulators and inhibitors. Thecontrol of angiogenesis has been found to be altered in certain diseasestates and, in many cases, the pathological damage associated with thedisease is related to the uncontrolled angiogenesis.

[0004] Both controlled and uncontrolled angiogenesis are thought toproceed in a similar manner. Endothelial cells and pericytes, surroundedby a basement membrane, form capillary blood vessels. Angiogenesisbegins with the erosion of the basement membrane by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a “sprout” offthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating the new blood vessel. In the disease state,prevention of angiogenesis could avert the damage caused by the invasionof the new microvascular system.

[0005] Persistent, unregulated angiogenesis occurs in a multiplicity ofdisease states, tumor metastasis and abnormal growth by endothelialcells and supports the pathological damage seen in these conditions. Thediverse pathological states created due to unregulated angiogenesis havebeen grouped together as angiogenic dependent or angiogenic associateddiseases. Therapies directed at control of the angiogenic processescould lead to the abrogation or mitigation of these diseases.

[0006] One example of a disease mediated by angiogenesis is ocularneovascular disease. This disease is characterized by invasion of newblood vessels into the structures of the eye such as the retina orcornea. It is the most common cause of blindness and is involved inapproximately twenty eye diseases. In age-related macular degeneration,the associated visual problems are caused by an ingrowth of chorioidalcapillaries through defects in Bruch's membrane with proliferation offibrovascular tissue beneath the retinal pigment epithelium. Angiogenicdamage is also associated with diabetic retinopathy, retinopathy ofprematurity, corneal graft rejection, neovascular glaucoma andretrolental fibroplasia. Other diseases associated with cornealneovascularization include, but are not limited to, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections, Kaposi sarcoma, Mooren ulcer, Terrien's marginaldegeneration, mariginal keratolysis, rheumatoid arthritis, systemiclupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis, Steven'sJohnson disease, periphigoid radial keratotomy, and corneal graphrejection.

[0007] Diseases associated with retinal/choroidal neovascularizationinclude, but are not limited to, diabetic retinopathy, maculardegeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthomaelasticum, Pagets disease, vein occlusion, artery occlusion, carotidobstructive disease, chronic uveitis/vitritis, mycobacterial infections,Lyme's disease, systemic lupus erythematosis, retinopathy ofprematurity, Eales disease, Bechets disease, infections causing aretinitis or choroiditis, presumed ocular histoplasmosis, Bests disease,myopia, optic pits, Stargarts disease, pars planitis, chronic retinaldetachment, hyperviscosity syndromes, toxoplasmosis, trauma andpost-laser complications. Other diseases include, but are not limitedto, diseases associated with rubeosis (neovasculariation of the angle)and diseases caused by the abnormal proliferation of fibrovascular orfibrous tissue including all forms of proliferative vitreoretinopathy.

[0008] Another disease in which angiogenesis is believed to be involvedis rheumatoid arthritis. The blood vessels in the synovial lining of thejoints undergo angiogenesis. In addition to forming new vascularnetworks, the endothelial cells release factors and reactive oxygenspecies that lead to pannus growth and cartilage destruction. Thefactors involved in angiogenesis may actively contribute to, and helpmaintain, the chronically inflamed state of rheumatoid arthritis.

[0009] Factors associated with angiogenesis may also have a role inosteoarthritis. The activation of the chondrocytes by angiogenic-relatedfactors contributes to the destruction of the joint. At a later stage,the angiogenic factors would promote new bone formation. Therapeuticintervention that prevents the bone destruction could halt the progressof the disease and provide relief for persons suffering with arthritis.

[0010] Chronic inflammation may also involve pathological angiogenesis.Such disease states as ulcerative colitis and Crohn's disease showhistological changes with the ingrowth of new blood vessels into theinflamed tissues. Bartonellosis, a bacterial infection found in SouthAmerica, can result in a chronic stage that is characterized byproliferation of vascular endothelial cells. Another pathological roleassociated with angiogenesis is found in atherosclerosis. The plaquesformed within the lumen of blood vessels have been shown to haveangiogenic stimulatory activity.

[0011] One of the most frequent angiogenic diseases of childhood is thehemangioma. In most cases, the tumors are benign and regress withoutintervention. In more severe cases, the tumors progress to largecavernous and infiltrative forms and create clinical complications.Systemic forms of hemangiomas, the hemangiomatoses, have a highmortality rate. Therapy-resistant hemangiomas exist that cannot betreated with therapeutics currently in use.

[0012] Angiogenesis is also responsible for damage found in hereditarydiseases such as Osler-Weber-Rendu disease, or hereditary hemorrhagictelangiectasia. This is an inherited disease characterized by multiplesmall angiomas, tumors of blood or lymph vessels. The angiomas are foundin the skin and mucous membranes, often accompanied by epistaxis(nosebleeds) or gastrointestinal bleeding and sometimes with pulmonaryor hepatic arteriovenous fistula.

[0013] Angiogenesis is prominent in solid tumor formation andmetastasis. Angiogenic factors have been found associated with severalsolid tumors such as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma,neuroblastoma, and osteosarcoma. A tumor cannot expand without a bloodsupply to provide nutrients and remove cellular wastes. Tumors in whichangiogenesis is important include solid tumors, and benign tumors suchas acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas.Prevention of angiogenesis could halt the growth of these tumors and theresultant damage to the animal due to the presence of the tumor.

[0014] It should be noted that angiogenesis has been associated withblood-born tumors such as leukemias, any of various acute or chronicneoplastic diseases of the bone marrow in which unrestrainedproliferation of white blood cells occurs, usually accompanied byanemia, impaired blood clotting, and enlargement of the lymph nodes,liver, and spleen. It is believed that angiogenesis plays a role in theabnormalities in the bone marrow that give rise to leukemia-like tumors.

[0015] Angiogenesis is important in two stages of tumor metastasis. Thefirst stage where angiogenesis stimulation is important is in thevascularization of the tumor which allows tumor cells to enter the bloodstream and to circulate throughout the body. After the tumor cells haveleft the primary site, and have settled into the secondary, metastasissite, angiogenesis must occur before the new tumor can grow and expand.Therefore, prevention of angiogenesis could lead to the prevention ofmetastasis of tumors and possibly contain the neoplastic growth at theprimary site.

[0016] Knowledge of the role of angiogenesis in the maintenance andmetastasis of tumors has led to a prognostic indicator for breastcancer. The amount of neovascularization found in the primary tumor wasdetermined by counting the microvessel density in the area of the mostintense neovascularization in invasive breast carcinoma. A high level ofmicrovessel density was found to correlate with tumor recurrence.Control of angiogenesis by therapeutic means could possibly lead tocessation of the recurrence of the tumors.

[0017] Angiogenesis is also involved in normal physiological processessuch as reproduction and wound healing. Angiogenesis is an importantstep in ovulation and also in implantation of the blastula afterfertilization. Prevention of angiogenesis could be used to induceamenorrhea, to block ovulation or to prevent implantation by theblastula.

[0018] In wound healing, excessive repair or fibroplasia can be adetrimental side effect of surgical procedures and may be caused orexacerbated by angiogenesis. Adhesions are a frequent complication ofsurgery and lead to problems such as small bowel obstruction.

[0019] Several kinds of compounds have been used to preventangiogenesis. Taylor et al. have used protamine to inhibit angiogenesis,see Taylor et al., Nature 297:307 (1982). The toxicity of protaminelimits its practical use as a therapeutic. Folkman et al. have disclosedthe use of heparin and steroids to control angiogenesis. See Folkman etal., Science 221:719 (1983) and U.S. Pat. Nos. 5,001,116 and 4,994,443.Steroids, such as tetrahydrocortisol, which lack gluco and mineralcorticoid activity, have been found to be angiogenic inhibitors.

[0020] Other factors found endogenously in animals, such as a 4 kDaglycoprotein from bovine vitreous humor and a cartilage derived factor,have been used to inhibit angiogenesis. Cellular factors such asinterferon inhibit angiogenesis. For example, interferon a or humaninterferon β has been shown to inhibit tumor-induced angiogenesis inmouse dermis stimulated by human neoplastic cells. Interferon β is alsoa potent inhibitor of angiogenesis induced by allogeneic spleen cells.See Sidky et al., Cancer Research 47:5155-5161 (1987). Human recombinanta interferon (alpha/A) was reported to be successfully used in thetreatment of pulmonary hemangiomatosis, an angiogenesis-induced disease.See White et al., New England J. Med. 320:1197-1200 (1989).

[0021] Other agents which have been used to inhibit angiogenesis includeascorbic acid ethers and related compounds. See Japanese Kokai TokkyoKoho No. 58-131978. Sulfated polysaccharide DS 4152 also showsangiogenic inhibition. See Japanese Kokai Tokkyo Koho No. 63-119500. Afungal product, fumagillin, is a potent angiostatic agent in vitro. Thecompound is toxic in vivo, but a synthetic derivative, AGM 12470, hasbeen used in vivo to treat collagen II arthritis. Fumagillin andO-substituted fumagillin derivatives are disclosed in EPO PublicationNos. 0325199A2 and 0357061A1.

[0022] PCT Application No. WO 92/14455 to Kaplan et al. is directed to amethod for controlling abnormal concentration of TNF-a by administeringthalidomide or thalidomide derivatives to a patient with toxicconcentrations of TNF-a.

[0023] The above compounds are either topical or injectabletherapeutics. Therefore, there are drawbacks to their use as a generalangiogenic inhibitor and lack adequate potency. For example, inprevention of excessive wound healing, surgery on internal body organsinvolves incisions in various structures contained within the bodycavities. These wounds are not accessible to local applications ofangiogenic inhibitors. Local delivery systems also involve frequentdressings which are impracticable for internal wounds, and increase therisk of infection or damage to delicate granulation tissue for surfacewounds.

[0024] Thus, a method and composition are needed that are capable ofinhibiting angiogenesis and which are easily administered. A simple andefficacious method of treatment would be through the oral route. If anangiogenic inhibitor could be given by an oral route, the many kinds ofdiseases discussed above, and other angiogenic dependent pathologies,could be treated easily. The optimal dosage could be distributed in aform that the patient could self-administer.

SUMMARY OF THE INVENTION

[0025] In accordance with the present invention, compositions andmethods are provided that are effective in inhibiting unwantedangiogenesis. These compositions are easily administered by differentroutes including oral and can be given in dosages that are safe andprovide angiogenic inhibition at internal sites. The present inventionprovides a method of treating mammalian diseases mediated by undesiredand uncontrolled angiogenesis by administering a composition comprisingan anti-angiogenic compound in a dosage sufficient to inhibitangiogenesis.

[0026] The present invention also includes angiogenic inhibitingcompounds that contain an epoxide group. These angiogenic inhibitingcompounds can be administered to a human or animal alone or with epoxidehydrolase inhibiting compounds.

[0027] The present invention is especially useful for treating certainocular neovascular diseases such as macular degeneration. The compoundswhich are contemplated as part of the present invention preferably canbe given orally to the patient and thereby halt the progression of thedisease. Other disease that can be treated using the present inventionare diabetic retinopathy, neovascular glaucoma and retrolentalfibroplasia.

[0028] Accordingly, it is an object of the present invention to providea compound and method to inhibit unwanted angiogenesis in a human oranimal.

[0029] It is yet another object of the present invention to provide acomposition of inhibiting angiogenesis by oral administration of thecomposition.

[0030] It is another object of the present invention to provide atreatment for diseases mediated by angiogenesis.

[0031] It is yet another object of the present invention to provide atreatment for macular degeneration.

[0032] It is yet another object of the present invention to provide atreatment for all forms of proliferative vitreoretinopathy includingthose forms not associated with diabetes.

[0033] It is yet another object of the present invention to provide atreatment for solid tumors.

[0034] It is yet another object of the present invention to provide amethod and composition for the treatment of blood-born tumors such asleukemia.

[0035] It is another object of the present invention to provide a methodand composition for the treatment of hemangioma.

[0036] It is another object of the present invention to provide a methodand composition for the treatment of retrolental fibroplasia.

[0037] It is another object of the present invention to provide a methodand composition for the treatment of psoriasis.

[0038] It is another object of the present invention to provide a methodand composition for the treatment of Kaposi's sarcoma.

[0039] It is another object of the present invention to provide a methodand composition for the treatment of Crohn's diseases.

[0040] It is another object of the present invention to provide a methodand composition for the treatment of diabetic retinopathy.

[0041] Other features and advantages of the invention will be apparentfrom the following description of preferred embodiments thereof.

[0042] These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

[0043]FIGS. 1 through 3 are a listing of representative compounds in thegenus represented by the following general formulas:

[0044]FIG. 4 is a listing of representative compounds in the genusrepresented by the following general formula:

[0045]FIG. 5 is a listing of representative compounds in the genusrepresented by the following general formula:

[0046]FIG. 6 shows the effect of thalidomide and EM12 on angiogenesis ina rabbit cornea model of angiogenesis.

[0047]FIG. 7 shows the effect of thalidomide on the area of cornealvascularization in a rabbit cornea model of angiogenesis.

DETAILED DESCRIPTION

[0048] The present invention includes compositions and methods for thetreatment of diseases that are mediated by angiogenesis. One embodimentof the present invention is the use of thalidomide or the metabolites ofthalidomide as disclosed herein to inhibit unwanted angiogenesis. Thepresent invention also includes compounds which cause dysmelia is thedeveloping fetus and have anti-angiogenic activity. The presentinvention comprises a method of treating undesired angiogenesis in ahuman or animal comprising the steps of administering to the human oranimal with the undesired angiogenesis a composition comprising aneffective amount of a teratogenic compound that is anti-angiogenic.

[0049] Compounds that can be used in accordance with the presentinvention include compounds included in the following general formulae.Examples of compounds that have anti-angiogenic properties having one ofthe following three formulae (A), (B), or (C):

[0050] In the above formulae A), B), and C), R₁, R₂, R₃ and R₄ can beselected from: —H; —OH;=O, straight chained and branched alkanes,alkenes, alkynes; cyclic alkanes, alkenes, and alkynes; combinations ofcyclic and acyclic alkanes, alkenes, and alkynes; alcohol, aldehyde,ketone, carboxylic acid, ester, or ether moieties in combination withacyclic, cyclic, or combination acyclic/cyclic moieties; aza; amino;—XO_(n) or —O—XO_(n), [where X═N and n=2; X═S and n=2 or 3; or X═P andn=1−3]; and halogens; R₅, R₆, R₇, and R₈ are each independently selectedfrom:

[0051] or —O— where Y is optional and is the same as defined above forR₁; and R₁₀ is the same as defined above for R₁, or (where Y is absent)R₁₀ is=O; and R₉ is a moiety having formula D), E), F), G) or H):

[0052] where each of R₁₁-R₁₇ is (independently) the same as definedabove for R₅;

[0053] where R₁₈, R₁₉ and R₂₀ are, independently selected from

[0054] and n=1 to 4.

[0055] Accordingly, another aspect of the present invention featuresinhibiting angiogenesis in a mammal by administering a therapeuticcomposition comprising one of the above-described compounds in a dosagesufficient to inhibit angiogenesis

[0056] In preferred embodiments, the compound has formula B), where R₅and R₆ are selected from the group consisting of:

[0057] and R₉ has formula F) or H); and R₁₄ and R₁₆ are selected fromthe group consisting of:

[0058] where R₂₁ is —H, —CH₃, or —OH. Specific preferred compoundsaccording to this aspect of the present invention include thalidomide,its precursors, metabolites and analogs. Particular analogs includeEM-12, N-phthaloyl-DL-glutamic acid (PGA) or N-phthaloyl-DL-glutamineanhydride. Examples of compounds that are members of this genus arelisted in FIGS. 1 through 3. It is to be understood that the compoundsincluded as part of the present invention are not to be limited to thosecompounds shown in FIGS. 1 through 3 and include all other compoundsthat are members of the genus described by the general formulas herein.

[0059] Compounds of the following formula that have anti-angiogenicproperties:

[0060] where R₂₂ and R₂₃ are (independently), —H, —F, —Cl, —Br, —I,—CH₃, or —CH₂—CH₃; and R₂₄ is —H, —CH₃, or —CH₂—CH₃.

[0061] The present invention also features inhibiting angiogenesis in amammal by administering a compound according to the above formulae in adosage sufficient to inhibit angiogenesis. Examples of specificcompounds that are members of this genus are listed in FIG. 4.

[0062] Angiogenesis inhibition hydrolysis products of thalidomide havingthe following general formula can be used in practicing the presentinvention:

[0063] where X is R₆ as defined above, or

[0064] and R₂₅ and R₂₆ are, independently, —OH, —H, or NH₂, and n=1through 4. Examples of such compounds are shown in FIG. 5.

[0065] Angiogenesis inhibition compounds having the following generalformula can be used in practicing the present invention:

[0066] wherein compounds of structure (1), wherein R is selected fromthe group consisting of hydrogen, alkyl radicals of 1 to 6 carbon atoms,the phenyl radical, and the benzyl radical; and wherein R′ is selectedfrom the group consisting of the phthalimido radical and the succinimidoradical and of structure (II), wherein X is CH₂ or C═O; R″ is H,—CH₂CH₃, —C₆H₅, —CH₂C₆H₅, —CH₂CH═CH₂, or (a) and hydrolysis products ofthe compounds wherein R″is H and the piperidino ring or both thepiperidino and the imido ring are hydrolyzed.

[0067] Another set of compounds that are considered part of the presentinvention are the epoxides of thalidomide, EM-12 and EM-138.Representative epoxide compounds are shown as follows:

[0068] It should be understood that the epoxide can be attached at the6,1 site on the benzene ring, the 1,2 site, the 2,3 site 3,4 or the 4,5site. All of these compounds are contemplated as part of the presentinvention.

[0069] The epoxides of the thalidomide, EM-12, and EM 138 can behydrolized to the following compounds:

[0070] It is to be understood that the hydroxyl group can be on carbons1, 2, 3, 4, 5 and 6 of the benzene ring. Also contemplated as part ofthe present invention are dihydroxyl compounds wherein the two hydroxylgroups are located bis to each other on carbons 1, 2, 3, 5 and 6 of theabove compounds. The epoxides, the hydrolysis products of the epoxides,and the hydrolysis products of the thalidomide are all contemplated tobe part of the present invention.

[0071] It is known that epoxides are hydrolized by a group of enzymesknown as epoxide hydrolases. There is a class of compounds which areepoxide hydrolase inhibitors. Examples of these compounds are valpromide(2-propylpentanamide) and valproic acid (2-propylpentanoic acid).Because epoxides are important angiogenesis inhibitors, it iscontemplated as part of the present invention, compositions comprisingany of the angiogenesis inhibitors compounds recited herein incombination with epoxide hydrolase inhibitors. The epoxide hydrolaseinhibitors can be administered to a human or animal together orsequentially. The expoxide group appears to be an important substituentcommon to several angiogenesis inhibitors. The use of epoxide hydrolaseinhibitors to potentiate the activity of any angiogenesis inhibitorcontaining an epoxide is contemplated as part of the present invention.For example, the epoxide hydrolase inhibitors can be administered withthe following epoxide-containing anti-angiogenesis compounds: AGM 1470,Eponimycin, microbial metabolites of Scolecobasidium arenariumdesignated f/2015, fr/111142 and fr/18487. See Oikawa, Biochem Biophys.Res. Comm, Vol. 81:1070 (1971) and Otsuka, J. Microbial. Biotech., Vol1:163 (1991).

[0072] It is contemplated as an embodiment of the present invention theuse of the epoxide containing angiogenesis inhibitors with or withoutepoxide hydrolase inhibitors as a treatment for diseases mediated byelevated or toxic levels of TNF-a. TNF-a has been recognized asmanifesting a dose dependent toxicity. If present at low levels for along period of time, TNF-a can result in cachexia. Cachexia is a generalweight loss and wasting occurring in the course of some chronic diseasessuch as cancer, opportunistic infections of AIDS, inflammatory diseases,parasitic diseases, tuberculosis, and high dose IL-2 therapy. Theepoxide containing angiogenesis inhibitors, with or without epoxidehydrolase inhibitors, are also effective in treating diseases such asseptic shock leprosy and graph vs. host disease.

[0073] Other embodiments are within the present invention. For example,other dysmelia-causing compounds can be used according to the presentinvention, e.g. 4-methylphthalic acid, pyridoxine, vasopressin,acetazolamide, or a compound having the following formula (where R═H,—OH, or —CH₃):

[0074] Other compounds which are teratogens, such as valproic acid(2-propylpentanoic acid), the retinoids, such as cis-retinoic acid, andrifampin may also be used in accordance with the invention.

[0075] In summary, the preferred compounds are thalidomide, as well asanalogs, hydrolysis products, metabolites and precursors of thalidomidethat are teratogenic, and, more specifically, that cause dismelia.However, it is to be understood that it is not necessary for a compoundto have both teratogenic activity and angiogenesis inhibiting activityto be considered part of the present invention. Dysmelia-causingcompounds can be identified by the general procedures of Helm,Arzneimittle forschung, 31(i/6):941-949 (1981), in which rabbit pups areexamined after exposure to the compound in utero. The compounds cangenerally be purchased, e.g., from Andrulis Pharmaceuticals, Beltsville,Md., or synthesized according to known procedures. It is to beunderstood that the compounds of the present invention can exist asenantiomers and that the racemic mixture of enantiomers or the isolatedenantiomers are all considered as within the scope of the presentinvention.

[0076] Many of the compounds that are contemplated as part of thepresent invention can be enriched in optically active enantiomers of thecompounds specified above. Specifically, Blaschke has reported that theS enanantiomers may be disproportionately responsible for thedismelia-producing effect of these compounds. See, generally Blaschke,Arzneimittelforschung 29:1640-1642 (1979). The above described articlesgenerally describe procedures to obtain optically active preparations ofthe compounds of interest. See, e.g. Shealy et al., Chem. Indus. 1030(1965); and Casini et al., Farmaco Ed. Sci. 19:563 (1964).

[0077] The compounds described above can be provided as pharmaceuticallyacceptable formulations using formulation methods known to those ofordinary skill in the art. These formulations can be administered bystandard routes. In general, the combinations may be administered by thetopical, transdermal, oral, rectal or parenteral (e.g., intravenous,subcutaneous or intramuscular) route. In addition, the combinations maybe incorporated into biodegradable polymers allowing for sustainedrelease of the compound, the polymers being implanted in the vicinity ofwhere drug delivery is desired, for example, at the site of a tumor. Thebiodegradable polymers and their use are described, for example, indetail in Brem et al., J. Neurosurg. 74:441-446 (1991).

[0078] The dosage of the compound will depend on the condition beingtreated, the particular compound, and other clinical factors such asweight and condition of the human or animal and the route ofadministration of the compound. It is to be understood that the presentinvention has application for both human and veterinary use. For oraladministration to humans, a dosage of between approximately 0.1 to 300mg/kg/day, preferably between approximately 0.5 and 50 mg/kg/day, andmost preferably between approximately 1 to 10 mg/g/day, is generallysufficient.

[0079] The formulations include those suitable for oral, rectal,ophthalmic, (including intravitreal or intracameral) nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intratracheal,and epidural) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by conventionalpharmaceutical techniques. Such techniques include the step of bringinginto association the active ingredient and the pharmaceutical carrier(s)or excipient(s). In general, the formulations are prepared by uniformlyand intimately bringing into associate the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0080] Formulations of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil emulsion and as a bolus, etc.

[0081] A tablet may be made by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface active ordispersing agent. Molded tablets may be made by molding, in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may be optionally coated or scored and maybe formulated so as to provide a slow or controlled release of theactive ingredient therein.

[0082] Formulations suitable for topical administration in the mouthinclude lozenges comprising the ingredients in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the ingredient to be administeredin a suitable liquid carrier.

[0083] Formulations suitable for topical administration to the skin maybe presented as ointments, creams, gels and pastes comprising theingredient to be administered in a pharmaceutical acceptable carrier. Apreferred topical delivery system is a transdermal patch containing theingredient to be administered.

[0084] Formulations for rectal administration may be presented as asuppository with a suitable base comprising, for example, cocoa butteror a salicylate.

[0085] Formulations suitable for nasal administration, wherein thecarrier is a solid, include a coarse powder having a particle size, forexample, in the range of 20 to 500 microns which is administered in themanner in which snuff is administered, i.e., by rapid inhalation throughthe nasal passage from a container of the powder held close up to thenose. Suitable formulations, wherein the carrier is a liquid, foradministration, as for example, a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient.

[0086] Formulations suitable for vaginal administration may be presentedas pessaries, tamports, creams, gels, pastes, foams or sprayformulations containing in addition to the active ingredient suchcarriers as are known in the art to be appropriate.

[0087] Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example, sealed ampules andvials, and may be stored in a freeze-dried (lyophilized) conditionsrequiring only the addition of the sterile liquid carrier, for example,water for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

[0088] Preferred unit dosage formulations are those containing a dailydose or unit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient.

[0089] It should be understood that in addition to the ingredients,particularly mentioned above, the formulations of the present inventionmay include other agents conventional in the art having regard to thetype of formulation in question, for example, those suitable for oraladministration may include flavoring agents.

[0090] Diseases associated with corneal neovascularization that can betreated according to the present invention include but are not limitedto, diabetic retinopathy, retinopathy of prematurity, corneal graftrejection, neovascular glaucoma and retrolental fibroplasia, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections, Kaposi sarcoma, Mooren ulcer, Terrien's marginaldegeneration, mariginal keratolysis, trauma, rheumatoid arthritis,systemic lupus, polyarteritis, Wegeners sarcoidosis, Scleritis, Steven'sJohnson disease, periphigoid radial keratotomy, and corneal graphrejection.

[0091] Diseases associated with retinal/choroidal neovascularizationthat can be treated according to the present invention include, but arenot limited to, diabetic retinopathy, macular degeneration, sickle cellanemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagets disease,vein occlusion, artery occlusion, carotid obstructive disease, chronicuveitis/vitritis, mycobacterial infections, Lyme's disease, systemiclupus erythematosis, retinopathy of prematurity, Eales disease, Bechetsdisease, infections causing a retinitis or choroiditis, presumed ocularhistoplasmosis, Bests disease, myopia, optic pits, Stargarts disease,pars planitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, trauma and post-laser complications. Other diseasesinclude, but are not limited to, diseases associated with rubeosis(neovasculariation of the angle) and diseases caused by the abnormalproliferation of fibrovascular or fibrous tissue including all forms ofproliferative vitreoretinopathy, whether or not associated withdiabetes.

[0092] Diseases associated with chronic inflammation can be treated bythe compositions and methods of the present invention. Diseases withsymptoms of chronic inflammation include inflammatory bowel diseasessuch as Crohn's disease and ulcerative colitis, psoriasis, sarcoidosisand rheumatoid arthritis. Angiogenesis is a key element that thesechronic inflammatory diseases have in common. The chronic inflammationdepends on continuous formation of capillary sprouts to maintain aninflux of inflammatory cells. The influx and presence of theinflammatory cells produce granulomas and thus, maintains the chronicinflammatory state. Inhibition of angiogenesis by the compositions andmethods of the present invention would prevent the formation of thegranulomas and alleviate the disease.

[0093] The compositions and methods of the present invention can be usedto treat patients with inflammatory bowel diseases such as Crohn'sdisease and ulcerative colitis. Both Crohn's disease and ulcerativecolitis are characterized by chronic inflammation and angiogenesis atvarious sites in the gastrointestinal tract. Crohn's disease ischaracterized by chronic granulomatous inflammation throughout thegastrointestinal tract consisting of new capillary sprouts surrounded bya cylinder of inflammatory cells. Prevention of angiogenesis by thecompositions and methods of the present invention inhibits the formationof the sprouts and prevents the formation of granulomas.

[0094] Crohn's disease occurs as a chronic transmural inflammatorydisease that most commonly affects the distal ileum and colon but mayalso occur in any part of the gastrointestinal tract from the mouth tothe anus and perianal area. Patients with Crohn's disease generally havechronic diarrhea associated with abdominal pain, fever, anorexia, weightloss and abdominal swelling. Ulcerative colitis is also a chronic,nonspecific, inflammatory and ulcerative disease arising in the colonicmucosa and is characterized by the presence of bloody diarrhea.

[0095] The inflammatory bowel diseases also show extraintestinalmanifestations such as skin lesions. Such lesions are characterized byinflammation and angiogenesis and can occur at many sites other than thegastrointestinal tract. The compositions and methods of the presentinvention are also capable of treating these lesions by preventing theangiogenesis, thus reducing the influx of inflammatory cells and thelesion formation.

[0096] Sarcoidosis is another chronic inflammatory disease that ischaracterized as a multisystem granulomatous disorder. The granulomas ofthis disease may form anywhere in the body and thus the symptoms dependon the site of the granulomas and whether the disease active. Thegranulomas are created by the angiogenic capillary sprouts providing aconstant supply of inflammatory cells.

[0097] The compositions and methods of the present invention can alsotreat the chronic inflammatory conditions associated with psoriasis.Psoriasis, a skin disease, is another chronic and recurrent disease thatis characterized by papules and plaques of various sizes. Prevention ofthe formation of the new blood vessels necessary to maintain thecharacteristic lesions leads to relief from the symptoms.

[0098] Another disease which can be treated according to the presentinvention is rheumatoid arthritis. Rheumatoid arthritis is a chronicinflammatory disease characterized by nonspecific inflammation of theperipheral joints. It is believed that the blood vessels in the synoviallining of the joints undergo angiogenesis. In addition to forming newvascular networks, the endothelial cells release factors and reactiveoxygen species that lead to pannus growth and cartilage destruction. Thefactors involved in angiogenesis may actively contribute to, and helpmaintain, the chronically inflamed state of rheumatoid arthritis.

[0099] Another disease that can be treated according to the presentinvention are hemangiomas, Osler-Weber-Rendu disease, or hereditaryhemorrhagic telangiectasia, solid or blood borne tumors and acquiredimmune deficiency syndrome.

[0100] This invention is further illustrated by the following examples,which are not to be construed in any way as imposing limitations uponthe scope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE I

[0101] The chick embryo chorioallantoic membrane assay described by Crumet al., Science 230:1375 et seq. (1985), is used to identify compoundsthat do not require further metabolic conversion. See also, U.S. Pat.No. 5,001,116, hereby incorporated by reference, which describes the CAMassay at col. 7 of the patent. Briefly, fertilized chick embryos areremoved from their shell on day 3 or 4, and a methylcellulose disccontaining the compound is implanted on the chorioallantoic membrane.The embryos are examined 48 hours later and, if a clear avascular zoneappears around the methylcellulose disc, the diameter of that zone ismeasured.

EXAMPLE II

[0102] Rabbit Cornea Angiogenesis Assay

[0103] Pellets for implantation into rabbit corneas were made by mixing110 μl of saline containing 12 μg of recombinant bFGF (TakedaPharmaceuticals-Japan) with 40 mg of sucralfate (Bukh Meditec-Denmark);this suspension was added to 80 μl of 12% hydron (Interferon Sciences)in ethanol. 10 μl aliquots of this mixture was then pipetted onto teflonpegs and allowed to dry producing approximately 17 pellets. A pellet wasimplanted into corneal micropockets of each eye of an anesthetizedfemale New Zealand white rabbit, 2 mm from the limbus followed bytopical application of erythromycin ointment onto the surface of thecornea. The animals were fed daily from 2 days post-implantation bygastric lavage with either drug suspended in 0.5% carboxymethylcellulose or 0.5% carboxymethyl cellulose alone. Thalidomide waspurchased from Andrulus Pharmaceutical (Maryland) and the EM-12 andSupidimide were kindly provided by Grunenthal GMBH (Germany). Theanimals were examined with a slit lamp every other day in a maskedmanner by the same corneal specialist. The area of cornealneovascularization was determined by measuring with a reticule thevessel length (L) from the limbus and the number of clock hours (C) oflimbus involved. A formula was used to determine the area of a circularband segment: C/12*3.1416 [r²−(r−L)²] where r=6 mm the measured radiusof the rabbit cornea. Various mathematical models were utilized todetermine the amount of vascularized cornea and this formula was foundto provide the most accurate approximation of the area of the band ofneovascularization that grows towards the pellet.

[0104] It is important to note that the rabbit cornea assay ispreferable because it will generally recognize compounds that areinactive per se but are metabolized to yield active compounds.Thalidomide related compounds, as shown below in Example III, are knownto be teratogens and are candidates for use in the present invention.

EXAMPLE III

[0105] Inhibition of bFGF Induced Corneal Neovascularization byThalidomide and Related Analog Expressed As Percent of Median Control onDay 8

[0106] Pellets containing bFGF and sucralfate were implanted intomicropockets of both corneas of rabbits according to Example II. Vesselingrowth into clear cornea from the limbus was first noted on day 2 andtreatments (200 mg/kg orally) were begun on this day. The area ofcorneal neovascularization was measured from day 4 through day 12. Day 8measurements were used for comparison between groups. No regression ofvessels and near maximal neovascularization was seen at this time point.Statistical analysis was performed with ANOVA with ranked data toaccount for interexperimental variation and to guard against anon-normal distribution of data (i.e. outliers) by utilizing anonparametric method.

[0107] The compounds tested were as follows:

[0108] Treatment with a dose of (200 mg/kg) of thalidomide resulted inan inhibition of the area of vascularized cornea that ranged from 30-51%in three experiments with a median inhibition of 36% (FIG. 6) (n=30eyes, p=0.0001, 2 way ANOVA with ranked data). The inhibition ofangiogenesis by thalidomide was seen after only two doses (FIG. 7). Therabbits did not demonstrate obvious sedation and there were no signs oftoxicity or weight loss. The teratogenic analog EM-12, which shares theother properties of thalidomide was also inhibitory, with a medianinhibition of 42% (n=10 eyes, p=0.002, 1-way ANOVA with ranked data).Supidimide, a nonteratogenic analog of thalidomide that retains thesedative properties of thalidomide, exhibited no activity (area 107% ofcontrol, n=10 eyes, not statistically different from control). Otheranalogs, PGA and PG acid displayed weaker inhibitory effects thanthalidomide (data not shown). The density of vessel ingrowth inthalidomide-treated animals was also markedly reduced.

EXAMPLE IV

[0109] EM-12 in Rabbit Cornea Assay

[0110] EM-12 was tested in the rabbit cornea assay described in ExampleII at 100 mg/kg/day and showed 21% inhibition, and at 200 mg/kg/day theassay showed 43% inhibition.

EXAMPLE V

[0111] Phthaloyl Glutamic Acid in CAM

[0112] Phthaloyl glutamic acid was tested in the above described CAMassay and exhibit an avascular zone with a mild scar.

EXAMPLE VI

[0113] Phthaloyl Glutamic Acid in Rabbit Cornea Assay

[0114] Phthaloyl glutamic acid described above at 200 mg/kg andexhibited 29% inhibition of angiogenesis.

EXAMPLE VIII

[0115] Phthaloyl Glutamic Anhydride in CAM Assay

[0116] Phthaloyl glutamic anhydride was test in the CAM assay describedabove and exhibited an avascular zone.

[0117] It should be understood, of course, that the foregoing relatesonly to preferred embodiments of the present invention and that numerousmodifications or alterations may be made therein without departing fromthe spirit and the scope of the invention as set forth in the appendedclaims.

1. A method of treating an eye condition associated with angiogenesis ina human or animal comprising administering to said human or animal anangiogenesis inhibiting amount of EM-138,


2. The method of claim 1 wherein the eye condition is selected from thegroup consisting of diabetic retinopathy, retinopathy of prematurity,corneal graft rejection, neovascular glaucoma, retrolental fibroplasia,epidemic keratoconjunctivitis, Vitamin A deficiency, contact lensoverwear, atopic keratitis, superior limbic keratitis, pterygiumkeratitis sicca, myopia, Terrien's marginal degeneration, mariginalkeratolysis, radial keratotomy, macular degeneration, post-lasercomplications, chronic retinal detachment; optic pits, hyperviscositysyndromes, chronic uveitis, chronic vitritis, ocular neovasculardisease, age-related macular degeneration, presumed ocularhistoplasmosis, infections causing retinitis or choroiditis,proliferative vitreoretinopathy, scleritis, Eales' disease, Best'sdisease, and trachoma.
 3. The method of claim 1 wherein the amountadministered is between approximately 0.1 and approximately 300mg/kg/day.
 4. The method of claim 3 wherein the amount administered isbetween approximately 0.5 and approximately 50 mg/kg/day.
 5. The methodof claim 4 wherein the amount administered is between approximately 1and approximately 10 mg/kg/day.
 6. The method of claim 1 wherein thecompound is administered in the form of a tablet or capsule.
 7. Themethod of claim 1 wherein the compound is administered in the form of alozenge, a cachet, a solution, a suspension, an emulsion, a powder, anaerosol, a suppository, a spray, a pastille, an ointment, a cream, apaste, a foam, a gel, a tamport, or a pessary.
 8. The method of claim 1wherein the administration is oral, parenteral, transdermal, or topical.9. The method of claim 1 wherein the administration is sublingual,buccal, rectal, vaginal, or nasal.
 10. A method of treating a non-tumorblood condition associated with angiogenesis in a human or animalcomprising administering to said human or animal an angiogenesisinhibiting amount of EM-138,


11. The method of claim 10 wherein the blood condition is selected fromthe group consisting of vein occlusion, artery occlusion, carotidobstructive disease, polyarteritis, atherosclerosis, Osler-Weber-Rendudisease, and sickle cell anemia.
 12. A method of treating an ulcerativedisease associated with angiogenesis in a human or animal comprisingadministering to said human or animal an angiogenesis inhibiting amountof EM-138,


13. The method of claim 12 wherein the ulcerative disease is selectedfrom the group consisting of bacterial ulcers, fungal ulcers, Mooren'sulcer, Wegener's sarcoidosis, Stevens-Johnson disease, Behcet's disease,pemphigoid, and ulceritive colitis.
 14. A method of treating a skincondition associated with angiogenesis in a human or animal comprisingadministering to said human or animal an angiogenesis inhibiting amountof EM-138,


15. The method of claim 14 wherein the skin condition is selected fromthe group consisting of acne, rosacea, chemical burns, and psoriasis.16. A method of treating an immune disease associated with angiogenesisin a human or animal comprising administering to said human or animal anangiogenesis inhibiting amount of EM-138,


17. The method of claim 16 wherein the immune disease is selected fromthe group consisting of rheumatoid arthritis, systemic lupus, acquiredimmune deficiency syndrome, and osteoarthritis.
 18. A method of treatingan infection associated with angiogenesis in a human or animalcomprising administering to said human or animal an angiogenesisinhibiting amount of EM-138,


19. The method of claim 18 wherein the infection is selected from thegroup consisting of Herpes simplex infections, Herpes zoster infections,Mycobacteria infections, protozoan infections, toxoplasmosis, syphilis,and Bartonellosis.
 20. A method for inhibiting undesired angiogenesis ina human or animal wherein the angiogenesis is associated with acondition selected from the group consisting of trauma, sjogren'ssyndrome, phylectenulosis, sarcoid, pseudoxanthoma elasticum,Stargardt's disease, Paget's disease, Lyme's disease, par planitis,pyogenic granulomas, and lipid degeneration comprising administering tothe human or animal an angiogenesis inhibiting amount of EM-138,


21. A method of inhibiting undesired angiogenesis in a human or animalwherein the angiogenesis is associated with a condition selected fromthe group consisting of Crohn's disease and chronic inflammationcomprising administering to the human or animal an angiogenesisinhibiting amount of EM-138,


22. A method of inhibiting undesired angiogenesis in a human or animalwherein the angiogenesis is associated with abnormal proliferation offibrovascular or fibrous tissue comprising administering to the human oranimal an angiogenesis inhibiting amount of EM-138,